Copper-catalysed ligation of azides and acetylenes

ABSTRACT

A copper catalyzed click chemistry ligation process is employed to bind azides and terminal acetylenes to provide 1,4-disubstituted 1,2,3-triazole triazoles. The process comprises contacting an organic azide and a terminal alkyne with a source of reactive Cu(I) ion for a time sufficient to form by cycloaddition a 1,4-disubstituted 1,2,3-triazole. The source of reactive Cu(I) ion can be, for example, a Cu(I) salt or copper metal. The process is preferably carried out in a solvent, such as an aqueous alcohol. Optionally, the process can be performed in a solvent that comprises a ligand for Cu(I) and an amine.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 12/804,644, filed onJul. 26, 2010, now U.S. Pat. No. 8,129,542, which is a continuation ofU.S. Serial No. 12/152,031, filed on May 9, 2008, now U.S. Pat. No.7,763,736, which is a continuation of U.S. Ser. No. 10/516,671, filed onMay 16, 2005, now U.S. Pat. No. 7,375,234, which is the National Stageof PCT/US2003/17311, filed on May 30, 2003, which claims the benefit ofU.S. Provisional Application Ser. No. 60/385,041, filed on May 30, 2002,each of which is incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support under Contract No. GM28384 by the National Institutes of Health and Contract No. CHE-9985553by the National Science Foundation. The U.S. government has certainrights in this invention.

FIELD OF THE INVENTION

The invention relates to a stepwise Huisgen cycloaddition processcatalyzed by copper(I). More particularly, the invention relates to acopper catalysed regioselective click chemistry ligation of azides andterminal alkynes to form triazoles.

BACKGROUND

Huisgen 1,3-dipolar cycloadditions are exergonic fusion processes thatunite two unsaturated reactants (R. Huisgen, in 1,3-DipolarCycloaddition Chemistry, (Ed.: A. Padwa), Wiley, New York, 1984, pp.1-176; and A. Padwa, in Comprehensive Organic Synthesis, (Ed.: B. M.Trost), Pergamon, Oxford, 1991, Vol. 4, pp 1069-1109). For a review ofasymmetric 1,3-dipolar cycloaddition reactions, see K. V. Gothelf, etal., Chem. Rev. 1998, 98, 863-909. For a review of syntheticapplications of 1,3-dipolar cycloadditions, see J. Mulzer, Org. Synth.Highlights 1991, 77-95. Huisgen 1,3-dipolar cycloadditions provide fastaccess to an enormous variety of 5-membered heterocycles (a) W.-Q. Fan,et al., in Comprehensive Heterocyclic Chemistry II, (Eds.: A. R.Katritzky, et al.), Pergamon, Oxford, 1996, Vol. 4, pp. 101-126; b) R.N. Butler, in Comprehensive Heterocyclic Chemistry II, (Eds.: A. R.Katritzky, et al.), Pergamon, Oxford, 1996, Vol. 4, pp 621-678; and c)K. Banert, Chem. Ber. 1989, 122, 911-918). The cycloaddition of azidesand alkynes to give triazoles is arguably the most useful member of thisfamily (a) R. Huisgen, Pure Appl. Chem. 1989, 61, 613-628; b) R.Huisgen, et al., Chem. Ber. 1967, 100, 2494-2507; c) W. Lwowski, in1,3-Dipolar Cycloaddition Chemistry, (Ed.: A. Padwa), Wiley, New York,1984; Vol. 1, Chapter 5; d) J. Bastide, et al., Bull. Soc. Chim. Fr.1973, 2555-2579; 2871-2887). However, probably because of concerns aboutthe safety of working with organic azides, synthetic chemists, in bothpure and applied fields, have not given this transformation the specialattention it deserves. Although the actual cycloaddition step may befaster and/or more regioselective for 1,3-dipoles other than azide, thelatter is by far the most convenient to introduce and to carry hiddenthrough many synthetic steps. Indeed, it appears to be the onlythree-atom dipole which is nearly devoid of side reactions.

Azides make only a fleeting appearances in organic synthesis, serving asone of the most reliable means to introduce a nitrogen substituent—R—X→[R—N₃]→R—NH₂. The azide intermediate is shown in brackets becauseit is generally reduced straightaway to the amine. Applications whichleverage the unique reactivity offered by the azide group itself aredisclosed by the following references from the laboratories of Aube,Banert, and Stoddart (a) P. Desai, et al., J. Am. Chem. Soc. 2000, 122,7226-7232; b) K. Banert, Targets in Heterocyclic Systems 1999, 3, 1-32;K. Banert, Liebigs Ann./Recl. 1997, 2005-18; c) J. Cao, et al., J. Org.Chem. 2000, 65, 1937-46 and references cited therein. Although azidechemistry can be hazardous, the hazard of working with these reagentsmay be minimized by employing appropriate safety precautions. Azides arechemically important as a crucial functional group for click chemistry(H. C. Kolb, et al., Angew. Chem. Int. Ed. 2001, 40, 2004-2021). Theuniqueness of azides for click chemistry purposes arises from theextraordinary stability of these reagents toward H₂O, O₂, and themajority of organic synthesis conditions. Indeed, organic azides,particularly in the aliphatic series, are exceptionally stable towardthe common reactive chemicals, ranging from dioxygen and water to theaqueous solutions of highly-functionalized organic molecules which makeup living cells. (E. Saxon, et al., Science 2000, 287, 2007-2010; and K.L. Kiick, et al., Proc. Natl. Acad. Sci. USA 2002, 99, 19-24). Thespring-loaded nature of the azide group remains invisible unless a gooddipolarophile is favorably presented.

In fact, it was the razor sharp reactivity window for this cycloadditionprocess which spawned our “in situ click chemistry” ideas—an approachwhich resulted in discovery of the most potent non-covalent inhibitor ofacetylcholinesterase known to date. (W. G. Lewis, et al., Angew. Chem.Int. Ed. 2002, 41, 1053-1057). However, even then the desiredtriazole-forming cycloaddition may require elevated temperatures and, inany case, usually results in a mixture of the 1,4- and 1,5-regioisomers(FIG. 1A), unless the acetylene component is attached to anelectron-withdrawing group such as a carbonyl or perfluoroalkyl (J.Bastide, et al., Bull. Chim. Soc. Fr. 1973, 2294-2296; N. P. Stepanova,et al., Zh. Org. Khim. 1985, 21, 979-983; N. P. Stepanova, et al., Zh.Org. Khim. 1989, 25, 1613-1618; and D. Clarke, et al., J. Chem. Soc.Perkin Trans. 1 1997, 1799-1804).

Efforts to control this 1,4-versus 1,5-regioselectivity problem have metwith varying success (P. Zanirato, J. Chem. Soc. Perkin Trans. I 1991,2789-2796; D. J. Hlasta, et al., J. Org. Chem. 1994, 59, 6184-6189; C.A. Booth, et al., Tet. Lett. 1998, 39, 6987-6990; S. J. Howell, et al.,Tetrahedron 2001, 57, 4945-4954; W. L. Mock, et al., J. Org. Chem.,1989, 54, 5302-5308; W. L. Mock Top. Curr. Chem. 1995, 175, 1-24; J.Chen, et al., Org. Lett. 2002, 4, 327-329; J. W. Wijnen, et al., Tet.Lett. 1995, 36, 5389-5392; M. P. Repasky, et al., Faraday Discuss. 1998,110, 379-389).

In one report, copper (I) catalyzed regiospecific synthesis ofpeptidotriazoles was achieved in organic solvents using free azides andterminal acetylenes attached to a solid support. (C. W. Tornøe, et al.,J. Org. Chem. 2002, 67, 3057). Reactants were non-equimolar. An earlierreport disclosed the formation, in the presence of copper (I), of atriazole, as a low yield byproduct, from a bifunctional reagent havingan acetylene group and an in situ generated azide (G. L'abbe, Bull. Soc.Chim. Belg. 1984, 93, 579-592).

SUMMARY OF THE INVENTION

One aspect of the invention is directed to a process for catalyzing aclick chemistry ligation reaction between a first reactant having aterminal alkyne moiety and second reactant having an azide moiety forforming a product having a triazole moiety. More particularly, the clickchemistry ligation reaction is catalyzed by an addition of Cu(II) in thepresence of a reducing agent for reducing said Cu(II) to Cu(I), in situ,in catalytic amount. Preferred reducing agents include ascorbate,metallic copper, quinone, hydroquinone, vitamin K₁, glutathione,cysteine, Fe²⁺, Co²⁺, and an applied electric potential. Furtherpreferred reducing agents include metals selected from the groupconsisting of Al, Be, Co, Cr, Fe, Mg, Mn, Ni, and Zn.

In an alternative aspect of the invention, a click chemistry ligationreaction between a first reactant having a terminal alkyne moiety andsecond reactant having an azide moiety for forming a product having atriazole moiety is catalyzed by performing the click chemistry ligationreaction in a solution in contact with metallic copper. The metalliccopper contributes directly or indirectly to the catalysis of the clickchemistry ligation reaction. In a preferred mode, the solution is anaqueous solution. The first and second reactants may be present duringthe click chemistry ligation reaction in equimolar amounts. Also, theclick chemistry ligation reaction may be performed in a solution incontact, at least in part, with a copper vessel.

In another aspect of the invention, a click chemistry ligation reactionbetween a first reactant having a terminal alkyne moiety and secondreactant having an azide moiety for forming a product having a triazolemoiety is catalyzed by an addition of a catalytic amount of a metal salthaving a metal ion selected from the group consisting of Au, Ag, Hg, Cd,Zr, Ru, Fe, Co, Pt, Pd, Ni, Rh, and W. In a preferred mode of thisaspect of the invention, the click chemistry ligation reaction isperformed in the presence of a reducing agent for reducing said metalion to a catalytically active form. Preferred reducing agents includeascorbate, quinone, hydroquinone, vitamin K₁, glutathione, cysteine,Fe²⁺, Co²⁺, an applied electric potential, and a metal selected from thegroup consisting of Al, Be, Co, Cr, Fe, Mg, Mn, Ni, and Zn.

In another aspect of the invention, a click chemistry ligation reactionbetween a first reactant having a terminal alkyne moiety and secondreactant having an azide moiety for forming a product having a triazolemoiety is performed in an aqueous solution and is catalyzed by acatalytic amount of copper (I). In a preferred mode of this aspect ofthe invention, the first and second reactants are present in equimolaramounts.

In another aspect of the invention, a click chemistry ligation reactionbetween a first reactant having a terminal alkyne moiety and secondreactant having an azide moiety for forming a product having a triazolemoiety is catalyzed by a catalytic amount of copper (I) while the firstand second reactants are present in equimolar amounts. In a preferredmode of this aspect of the invention, the click chemistry ligationreaction is performed in an aqueous solution.

In another aspect of the invention, a click chemistry ligation reactionbetween a first reactant having a terminal alkyne moiety and secondreactant having an azide moiety for forming a product having a triazolemoiety is performed in a solution containing a catalytic amount ofcopper (I). However, in this instance, there is a proviso that eitherthe first or second reactant is toxic or expensive and the remainingreactant is present in molar excess.

In another aspect of the invention, a click chemistry ligation reactionbetween a first reactant having a terminal alkyne moiety and secondreactant having an azide moiety for forming a product having a triazolemoiety is performed inside a cell. The cell is of a type that contains acatalytic quantity of copper(I) sufficient to catalyze the clickchemistry ligation reaction. The copper(I) contributes to a catalysis ofthe click chemistry ligation reaction.

In another aspect of the invention, a click chemistry ligation reactionbetween a first reactant having a terminal alkyne moiety and secondreactant having an azide moiety for forming a product having a triazolemoiety is performed in a solvent containing a catalytic amount of ametal ion. The metal ions are selected from the group of metalsconsisting of Cu, Au, Ag, Hg, Cd, Zr, Ru, Fe, Co, Pt, Pd, Ni, Rh, and W.The metal ion contributes directly or indirectly to a catalysis of theclick chemistry ligation reaction. The metal ion is coordinated to aligand for solubilizing such metal ion within the solvent, forinhibiting oxidation of such metal ion, and for dissociating, in wholeor in part, from such metal ion during the catalysis of the clickchemistry ligation reaction by said metal ion. A preferred ligand isacetonitrile. Another preferred ligand is a cyanide, nitrile, orisonitrile. Another preferred ligand is water. Other preferred ligandsinclude nitrile, isonitrile, primary, secondary, or tertiary amine, anitrogen bearing heterocycle, carboxylate, halide, alcohol, thiol,sulfide, phosphine, and phosphite. Other preferred ligands arepolyvalent and include one or more functional groups selected from thegroup consisting of nitrile, isonitrile, primary, secondary, or tertiaryamine, a nitrogen bearing heterocycle, carboxylate, halide, alcohol,thiol, sulfide, phosphine, and phosphite.

Another aspect of the invention is directed to a reactive intermediatefor producing a product having triazole moiety. The reactiveintermediate is represented by the following 6-membered ring structure:

In the above structure, R¹ is a 4-triazole substituent, R² is a1-triazole substituent, L is a Cu ligand, and “n” is 1, 2, or 3.

Another aspect of the invention is directed to a reactive intermediatefor producing a triazole. The reactive intermediate is represented bythe following 6-membered ring structure:

In the above structure, R¹ is employable as a 4-triazole substituent, R²is employable as a 1-triazole substituent, L is a Cu ligand, and “n” is1, 2, 3, or 4.

Another aspect of the invention is directed to a two step process ofderivatizing an amine containing molecule with a triazole. In the firststep of this process the amine containing molecule is derivatized toform a terminal alkyne. Then, the product of the first step is ligatedwith an azide containing molecule by addition of the azide containingmolecule in the presence of a catalytic amount of Cu to form a triazolederivative of the amine containing molecule.

Another aspect of the invention is directed to one step process forproducing a polyvalent triazole. In this process, a polyazide core isderivatized by addition of a molecule having a terminal alkyne in thepresence of a catalytic amount of Cu.

Another aspect of the invention is directed to a one step process forproducing a polyvalent triazole. In this process, a polyalkyne core isderivatized by addition of an azide containing molecule in the presenceof a catalytic amount of Cu.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates a prior art uncatalysed Huisgen cycloaddition ofazides and terminal alkynes.

FIG. 1B illustrates a copper catalysed regioselective ligation of azidesand terminal alkynes.

FIG. 2 illustrates proposed mechanism for the step-wise copper catalysedligation of azides and terminal alkynes and compares this mechanism witha direct cycloaddition.

FIGS. 3A and 3B illustrate a table showing the synthesis of1,4-disubstituted[1,2,3]-triazoles catalyzed by Cu^(I) in the presenceof ascorbate. All reactions were carried out in water with t-butanol asco-solvent, 0.25-0.5M in reactants, with 1 mol % of CuSO₄ and 10 mol %of sodium ascorbate, and were complete in 12-24 hours.

FIG. 4 illustrates copper catalysed regioselective ligation of azidesand terminal alkynes employing Cu(0) as a reducing agent to reduce Cu(2)to Cu(1).

FIG. 5 illustrates use of human plasma as a solvent for the reaction. At100 mM azide and 200 mM acetylene, the reaction is complete in 12-14hours. At 2 mM azide and 4 mM acetylene, the reaction is approximately80% complete after 48 hours. Note that the reaction does not decline inplasma, but merely slows down. Despite the high concentration ofproteins and the binding of Cu to protein, the reaction proceeds well.

FIG. 6 illustrates an exemplary two step procedure for derivatizingamine containing molecules, e.g. erythromycin, with triazoles. Theprocedure is applicable to any amine containing molecule.

FIG. 7 illustrates a one step process using Cu catalysis forderivatizing polyazide cores to produce polyvalent triazoles.

FIG. 8 illustrates a one step process using Cu catalysis forderivatizing polyalkyne cores to produce polyvalent triazoles.

DETAILED DESCRIPTION

The process is experimentally simple and appears to have enormous scope.While a number of copper(I) sources can be used directly (vide infra),it is disclosed that the catalyst is better prepared in situ byreduction of Cu^(II) salts, which are less costly and often purer thanCu^(I) salts (CuSO₄.5H₂O serves well). As the reductant, ascorbic acidand/or sodium ascorbate proved to be excellent, for they allowpreparation of a broad spectrum of 1,4-triazole products in high yieldsand purity at 0.25-2 mol % catalyst loading. For a review of reactionsof L-ascorbic acid with transition metals see M. B. Davies Polyhedron1992, 11, 285-321 and references cited therein; redox properties ofascorbic acid are summarized in C. Creutz Inorg. Chem. 1981, 20, 4449.The reaction appears to be very forgiving and does not require anyspecial precautions. It proceeds to completion in 6 to 36 hours atambient temperature in a variety of solvents, including aqueoust-butanol or ethanol and, very importantly, water with no organicco-solvent. Starting materials do not need to be dissolved in thereaction solvent. The reaction seems to proceed just as efficiently aslong as adequate stirring is maintained. Although most experiments wereperformed at near neutral pH, the catalysis seems to proceed well at pHvalues ranging from ca. 4 to 12. The catalytic process is very robustand insensitive to usual reaction parameters.

It is further disclosed that Cu⁰ can also be used as a source of thecatalytic species. Although these reactions may take longer to proceedto completion, the experimental procedure is exceedingly simple. Forexample, bis-triazole shown in entry 2 (FIG. 3A) was obtained inquantitative yield after stirring the corresponding azide and acetylenecomponents for 24 h with ca. 2 g of copper metal turnings. The turningswere removed at the end of the reaction, and the pure white product wascollected by simple filtration.

The reaction between phenyl propargyl ether and benzylazide in thepresence of 5 mol % of sodium ascorbate and 1 mol % of copper(II)sulfate in 2:1 water/t-butanol mixture furnished the 1,4-disubstitutedtriazole product in 91% yield after stirring for 8 hours at roomtemperature in a capped scintillation vial, but otherwise with no effortto exclude oxygen [eq. (2)]. The regiochemistry of the product wasestablished by NOE experiments and confirmed by the X-raycrystallographic analysis.

For comparison, the thermal reaction (neat, 92° C., 18 h) between thesesubstrates gives both regioisomers in a ratio of 1.6:1 in favor of the1,4-isomer, as illustrated in FIG. 1A.

The scope of this copper-catalyzed triazole synthesis is partly revealedby the examples in FIGS. 3A and B; note especially the lack offunctional group interference. These triazoles are obtained using aprocedure which generally involves little more than stirring thereagents and filtering off pure products. Variously substituted primary,secondary, tertiary, and aromatic azides readily participate in thistransformation. Tolerance for variations in the acetylene component isalso excellent.

Copper(I) salts, for example CuI, CuOTf.C₆H₆ and [Cu(NCCH₃)₄]PF₆, canalso be used directly in the absence of a reducing agent. Thesereactions usually require acetonitrile as co-solvent and one equivalentof a nitrogen base (e.g. 2,6-lutidine, triethylamine,diisopropylethylamine, or pyridine). However, formation of undesiredbyproducts, primarily diacetylenes, bis-triazoles, and5-hydroxytriazoles, was often observed. For a recent summary of thereactions of copper(I) complexes with dioxygen, see S. Schindler Eur. J.Inorg. Chem. 2000, 2311-2326 and A. G. Blackman, W. B. Tolman inStructure and Bonding, B. Meunier, Ed., Springer-Verlag, Berlin,Heidelberg, 2000, vol. 97, p. 179-211. This complication with direct useof Cu^(I)-species was minimized when 2,6-lutidine was used, andexclusion of oxygen further improved product purity and yield. Forexample, ethylpropiolate and benzyl azide furnished the corresponding1,4-triazole in 55% yield when this procedure was used, but only traceamount of the product was obtained with 1 equiv. of triethylamine and noexclusion of oxygen. Even though a broad range of both acetylene andazide components react readily in the acetonitrile system, we prefer theeven more reliable and simple Cu^(II)/ascorbate aqueous system (with orwithout co-solvents and amine buffers/additives).

A mechanistic proposal for the catalytic cycle is illustrated in FIG. 2.It begins unexceptionally with formation of the copper(I) acetylide i(G. van Koten, J. G. Noltes in Comprehensive Organometallic Chemistry,G. Wilkinson, Ed., vol. 2, chap. 14, Pergamon Press, 1982, 720). Asexpected, no reaction is observed with internal alkynes. It is disclosedherein that extensive density functional theory calculations offercompelling evidence which strongly disfavors, by approximately 12-15kcal, the concerted [2+3] cycloaddition (B-direct) and points to astepwise, annealing sequence (B1→B2→B3), which proceeds via the6-membered copper containing intermediate iii (M. P. Doyle, et al., inModern Catalytic Methods for Organic Synthesis with Diazo CompoundsWiley (New York), 1997, 163-248). Hence, the term ‘ligation’ is employedherein to denote the step-wise [2+3] cycloaddition catalyzed bycopper(I).

The Cu^(I)-catalyzed transformation described here—a high-yielding andsimple to perform ‘fusion’ process leading to a thermally andhydrolytically stable triazole connection—is an ideal addition to thefamily of click reactions. The process exhibits broad scope and provides1,4-disubstituted[1,2,3]-triazole products in excellent yields and nearperfect regioselectivity. The reactivity of copper(I) acetylides withorganic azides is disclosed herein to be effectively unstoppable.

This new catalytic process offers an unprecedented level of selectivity,reliability and scope for those organic synthesis endeavors which dependon the creation of covalent links between diverse building blocks.Several applied projects which highlight the capabilities of the processare illustrated in FIGS. 6-8.

Experimental Procedure General procedure as exemplified for thesynthesis of17-[1-(2,3-dihydroxypropyl)-1H-[1,2,3]triazol-4-yl]-estradiol

17-ethynyl estradiol (888 mg, 3 mmol) and (S)-3-azidopropane-1,2-diol(352 mg, 3 mmol) were suspended in 12 mL of 1:1 water/t-butanol mixture.Sodium ascorbate (0.3 mmol, 300 μL of freshly prepared 1M solution inwater) was added, followed by copper(II) sulfate pentahydrate (7.5 mg,0.03 mmol, in 100 μL of water). The heterogeneous mixture was stirredvigorously overnight, at which point it cleared and TLC analysisindicated complete consumption of the reactants. The reaction mixturewas diluted with 50 mL of water, cooled in ice, and the whiteprecipitate was collected by filtration. After washing with cold water(2×25 mL), the precipitate was dried under vacuum to afford 1.17 g (94%)of pure product as off-white powder. M.p. 228-230° C. Elemental analysiscalculated: C, 64.02%; H, 7.71%; N, 9.74%. found: C, 64.06%; H, 7.36%;N, 9.64%.

Reducing Environment Effect:

Cu(I) is very easily oxidized to Cu(II)—primarily by oxygen, but evenmilder oxidants can accomplish this. Additionally, Cu(I) mediatesoxidative coupling of acetylenes and other organocopper species, whichleads to reduced yields and contaminated products. All these problemscan be circumvented by the use of a mild reducing agent. Variousreducing agents can be used: ascorbate, hydroquinone, other quinones(such as vitamin K₁), copper turnings/wire, glutathione, cysteine, Fe²⁺,Co²⁺, etc. Virtually any reductant may be employed that is not sopowerful so as to rapidly reduce Cu(II) to Cu(0).

Ligands: Acetonitrile Effect:

Metals do not exist in solutions “naked”—there are always ligandspresent in the coordination sphere, be it solvent molecules or‘designer’ ligands. Ligands have a profound effect on reactivity of themetal center, as well as red/ox properties of the metal: (a) they canstabilize a certain oxidation state of the metal, e.g. Cu(I) is thedesirable form in our case, and (b) just as importantly, they can keepthe catalytic species in the solution, thereby making it constantlyavailable for the catalysis. Both of these requirements have to befulfilled in order for a metal/ligand combination to be useful incatalyzing a desired transformation cycloaddition, or ligation, in thepresent instance.

Copper-mediated organic transformations have been studied for over 70years, and the literature on the subject is quite extensive. Animportant lesson of the prior art is that cyanides and/or nitriles aresome of the best ligands for Cu(I), which is usually tetracoordinated,forming tetrahedral complexes. In fact, acetonitrile coordinates toCu(I) so strongly that [Cu(CH₃CN)₄]⁺PF6⁻ complex is a commerciallyavailable, oxygen-insensitive Cu(I) preparation (i.e., this Cu(I) isunreactive). This ‘overstabilization’ is clearly a liability whenreactivity is our goal. The reaction indicated below illustrates thepoint. When water/alcohol mixtures are employed as solvents (note thatboth are weak ligands for Cu(I)), the reaction is complete in under 6hrs. However, when acetonitrile is used as a solvent, no reaction isobserved at all even after 24 hrs under otherwise identical conditions.

To explain this phenomenon, recall the mechanism of the reaction. Inorder for the ligation to proceed, the azide must coordinate to thecopper (step B1) and one ligand must be removed from the coordinationsphere of the metal, and in case of a strongly coordinated acetonitrile,this step is disfavored.

Therefore, in order to have a useful reactivity window, one shouldchoose or design such ligands that do bind to the metal relatively well,keep it in the correct oxidation state and in solution (i.e. notaggregated to the point of forming a polymeric precipitate), but in thesame time can come off the metal center to allow formation ofintermediate ii, which is a necessary step in the catalytic sequence. Touse the example in hand, addition of an amine, such as triethylamine or2,6-lutidine to the acetonitrile system described above, solves theproblem of reactivity—the product is formed in quantitative yield afterca. 8 hrs.

Preferred ligands include cyanides, nitriles, isonitriles, primary orsecondary amines, carboxylates, halides, alcohols, and thiols. Chlorideis the preferred halide and best employed at 1-5 M. Polyvalent ligandsthat include one or more functional groups selected from nitrile,isonitrile, primary or secondary amine, carboxylate, alcohol, and thiolmay also be employed.

Other Metal Catalysts:

Cu is not the only metal capable of catalyzing this type ofcycloaddition. As long as intermediate ii can be formed (i.e. the metalis or can become coordinatively unsaturated), other metals known to formstable acetylides may also be employed. Exemplary metals that can formstable acetylides include Cu, Au, Ag, Hg, Cd, Zr, Ru, Fe, Co, Pt, Pd,Ni, Rh, and W. It is a matter of finding the right metal/ligandcombination. Copper is unique among other metals because it is so wellsupported in aqueous systems, which results in exceedingly simpleexperimental procedure and amazing functional group tolerance.

Catalysis of Ligation Reaction by Metallic Container:

Metallic containers can also be used as a source of the catalyticspecies to catalyze the ligation reaction. For example, a coppercontainer)(Cu⁰) may be employed to catalyze the reaction. In order tosupply the necessary ions, the reaction solution must make physicalcontact with the a copper surface of the container. Alternatively, thereaction may be run in a non-metallic container, and the catalytic metalions supplied by contacting the reaction solution with a copper wire,copper shavings, or other structures. Although these reactions may takelonger to proceed to completion, the experimental procedure isexceedingly simple. For example, bis-triazole shown in entry 2 (FIG. 3A)was obtained in quantitative yield after stirring the correspondingazide and acetylene components for 24 h with ca. 2 g of copper metalturnings. The turnings were removed at the end of the reaction, and thepure white product was collected by simple filtration.

Alternative Reducing Agents:

Metals may be employed as reducing agents to maintain the oxidationstate of the Cu (I) catalyst or of other metal catalysts. Preferredmetallic reducing agents include Cu, Al, Be, Co, Cr, Fe, Mg, Mn, Ni, andZn. Alternatively, an applied electric potential may be employed tomaintain the oxidation state of the catalyst.

In Vivo Catalysis:

The reaction proceeded well in fresh human plasma (protein loading 65-85mg/mL, C_(azide)=C_(alkyne)=5 mM; C_(Cu+)=100 mM) and in whole blood,indicating that copper species remained available for the catalysisdespite being heavily bound to plasma proteins and indicating that theligation reaction can be catalyzed by copper and/or other metals ionsand templates in vivo, including intracellularly. The reaction proceedsfresh human plasma and intracellularly in whole blood without noticeableloss of catalytic turnover and without noticeable protein precipitation.

Cu(I) Salt Used Directly:

If Cu(I) salt is used directly, no reducing agent is necessary, butacetonitrile or one of the other ligands indicate above should be usedas a solvent (to prevent rapid oxidation of Cu(I) to Cu(II) and oneequivalent of an amine should be added (to accelerate the otherwiseextremely slow reaction—vide supra). In this case, for better yields andproduct purity, oxygen should be excluded. Therefore, the ascorbate (orany other reducing) procedure is often preferred over the unreducedprocedure. The use of a reducing agent is procedurally simple, andfurnishes triazole products in excellent yields and of high purity.

What is claimed is:
 1. A copper catalyzed process for preparing a1,4-disubstituted 1,2,3-triazole comprising: contacting a solution of anorganic azide and a terminal alkyne with a source of catalytic Cu(I) ionfor a time sufficient to form by cycloaddition a 1,4-disubstituted1,2,3-triazole.
 2. The process of claim 1 wherein the solution furthercomprises at least one ligand comprising at least one functional groupselected from the group consisting of a nitrile, an isonitrile, aprimary amine, a secondary amine, a tertiary amine, a nitrogen bearingheterocycle, a carboxylate, a halide, an alcohol, a thiol, a sulfide, aphosphine, and a phosphite.
 3. The process of claim 1 wherein thesolution is an aqueous solution.
 4. A copper catalyzed process forpreparing a 1,4-disubstituted 1,2,3-triazole comprising: contacting asolution of an organic azide and a terminal alkyne with a source ofcatalytic Cu(I) ion for a time sufficient to form by cycloaddition a1,4-disubstituted 1,2,3-triazole; wherein the source of Cu(I) ioncomprises a Cu(I) salt.
 5. The process of claim 4 wherein the solutionfurther comprises at least one ligand comprising a functional groupselected from the group consisting of a nitrile, an isonitrile, aprimary amine, a secondary amine, a tertiary amine, a nitrogen bearingheterocycle, a carboxylate, a halide, an alcohol, a thiol, a sulfide, aphosphine, and a phosphite.
 6. The process of claim 4 wherein thesolution further comprises at least one ligand comprising a nitrogenbearing heterocycle group.
 7. The process of claim 4 wherein thesolution further comprises at least one polyvalent ligand comprising oneor more functional groups selected from the group consisting of anitrile, an isonitrile, a primary amine, a secondary amine, a tertiaryamine, a nitrogen bearing heterocycle, a carboxylate, a halide, analcohol, a thiol, a sulfide, a phosphine, and a phosphite.
 8. Theprocess of claim 4 wherein the solution further comprises at least onepolyvalent ligand comprising one or more nitrogen bearing heterocyclegroups.
 9. The process of claim 4 wherein the solution further comprisesat least one polyvalent ligand comprising one or more alcohol groups.10. The process of claim 4 wherein the Cu(I) salt is selected from thegroup consisting of CuI, CuOSO₂CH₃, and [Cu(CH₃CN)₄]PF₆.
 11. The processof claim 4 wherein the solution further comprises an amine.
 12. Theprocess of claim 4 wherein the solution is an aqueous solution.
 13. Theprocess of claim 12 wherein the aqueous solution further comprises analcohol.
 14. The process of claim 12 wherein the aqueous solution isfree from an organic co-solvent.
 15. A copper catalyzed process forpreparing a 1,4-disubstituted 1,2,3-triazole comprising: contacting asolution of an organic azide and a terminal alkyne with a source ofcatalytic Cu(I) ion for a time sufficient to form by cycloaddition a1,4-disubstituted 1,2,3-triazole; wherein the source of catalytic Cu(I)is Cu(II) ion in combination with a reducing agent.
 16. The process ofclaim 15 wherein the reducing agent comprises sodium ascorbate.
 17. Theprocess of claim 15 wherein the reducing agent comprises glutathione.18. The process of claim 15 wherein the reducing agent comprisescysteine.
 19. The process of claim 15 wherein the Cu(II) ion is providedas a Cu(II) salt.
 20. The process of claim 15 wherein the solution is anaqueous solution.
 21. The process of claim 20 wherein the aqueoussolution is free from an organic co-solvent.
 22. The process of claim 15wherein the solution comprises a primary amine buffer.
 23. The processof claim 15 wherein the solution further comprises a ligand for Cu(I)ion.
 24. The process of claim 23 wherein the ligand for Cu(I) ioncomprises one or more functional groups selected from the groupconsisting of a nitrile, an isonitrile, a primary amine, a secondaryamine, a tertiary amine, a nitrogen bearing heterocycle, a carboxylate,a halide, an alcohol, a thiol, a sulfide, a phosphine, and a phosphite.25. The process of claim 15 wherein the solution further comprises aligand for Cu(I) comprising one or more nitrogen bearing heterocyclegroups.
 26. The process of claim 15 wherein the solution furthercomprises at least one polyvalent ligand comprising one or more alcoholgroups.